A Computational Algorithm to Predict shRNA Potency

Abstract

The strength of conclusions drawn from RNAi-based studies is heavily influenced by the quality of tools used to elicit knockdown. Prior studies have developed algorithms to design siRNAs. However, to date, no established method has emerged to identify effective shRNAs, which have lower intracellular abundance than transfected siRNAs and undergo additional processing steps. We recently developed a multiplexed assay for identifying potent shRNAs and have used this method to generate ~250,000 shRNA efficacy data-points. Using these data, we developed shERWOOD, an algorithm capable of predicting, for any shRNA, the likelihood that it will elicit potent target knockdown. Combined with additional shRNA design strategies, shERWOOD allows the ab initio identification of potent shRNAs that target, specifically, the majority of each gene’s multiple transcripts. We have validated the performance of our shRNA designs using several orthogonal strategies and have constructed genome-wide collections of shRNAs for humans and mice based upon our approach.

COP1/DET1/ETS axis regulates ERK transcriptome and sensitivity to MAPK inhibitors

“…We generated A2058 cells expressing MAPK-ETV1 stability sensors consisting of fusion proteins of ETV1 fragments with the green fluorescent EGFP and the red fluorescent tdTomato, under the same MSCV retroviral promoter with an internal ribosomal entry site (IRES) ( Figure 3A) (28). We found that the 174-amino acid amino-terminal of ETV1, when fused to EGFP (EGFP-nETV1), was more robustly expressed than the full-length ETV1 (EGFP- To identify genes that regulate MAPK signaling-dependent ETV1 protein stability, we performed a pooled genome-wide RNAi screen using a miR-30-based shRNA library of approximately 76,000 hairpins targeting approximately 20,000 human genes (29,30). To obtain hits that maintained ETV1 protein levels despite MAPK pathway inhibition, we treated the A2058 MAPK-ETV1 sensor cells with vemurafenib for 24 hours prior to isolation of the cells with the highest and lowest 5% as well as cells with the middle 20% EGFP/tdTomato fluorescence ratio by FACS ( Figure 3E).…”

Next-generation libraries for robust RNA interference-based genome-wide screens

“…We previously developed an shRNA expression vector in which this CNNC motif is restored (5). A similar strategy has been reported by others (16). We also wanted to test a pair of point mutations in the shRNA loop region that would generate a restriction site in the encoding DNA ( Fig.…”

“…A major advance was the establishment of the socalled sensor assay, which enabled the massively parallel determination of shRNA activities (18). From data generated with the sensor assay, rules for shRNA activity were abstracted (18) and a proprietary algorithm for the prediction of shRNA activity was developed (16). Our goal was to test which rules are predictive of shRNA activity specifically in our expression context.…”

Network Architecture Predisposes an Enzyme to Either Pharmacologic or Genetic Targeting

“…Rightward shifts in knockdown/inhibitor discrepancy, which peaked at ~85% perturbation, were also observed with various other measures of MAPK signaling output (Figures S1C–S1E). 80+% knockdown efficiency is often assumed to be acceptable for functional studies (Knott et al, 2014). However, our results here suggested that this extent of targeting would be insufficient for disrupting a MAPK cascade with negative feedback, especially when compared with a MEK inhibitor.…”

“…If knockdown of an enzyme and pharmacologic competition for substrate are both ~100% effective, then these two perturbations should yield identical results, provided that the enzyme does not have a catalysis-independent function (Knight and Shokat, 2007). However, because knockdowns are often partial and small molecule doses are limited by pharmacokinetics and off-target toxicities, molecular genetics and pharmacology typically yield only a fractional inhibition in vivo (Bollag et al, 2010; Knott et al, 2014). Given a fractional perturbation, it is unclear whether knockdown and small molecule approaches are truly equal, especially in cascades that contain feedback, feedforward, and autoregulatory mechanisms.…”